We have shown in the fetus and newborn, that in pulmonary vascular relaxation induced by cGMP-elevating agents, cGMP-dependent Protein Kinase (PKG) plays a primary role. We have also reported that hypoxia attenuates nitric oxide-cGMP-mediated relaxation by decreasing PKG activity in fetal pulmonary vessels. In general, in pulmonary vascular smooth muscle (SMC), the nitric oxide-cGMP-PKG pathway leads to activation of myosin light chain phosphatase (MLCP) and relaxation whereas the RhoA-Rho-Kinase (ROCK) pathway promotes contraction, but the interactions between these pathways are complex. Based on our preliminary data, we hypothesize that PKG is the master molecule in fetal pulmonary vascular smooth muscle that regulates interactions among RhoA, Rho Kinase and MLCP, to regulate vasoreactivity. This novel and new hypothesis will be explored in this proposal. In isolated intrapulmonary arteries and veins and in vascular smooth muscle cells cultured from these vessels, we will first test the hypothesis that hypoxia-induced decrease in pulmonary vascular relaxation is in part due to alteration in PKG function and altered interactions among PKG, RhoA-ROCK and MYPT1 subunit of MLCP resulting in decreased activation of MLCP and decreased relaxation. We will also test the hypothesis that these alterations in PKG interactions with RhoA-Rock and MYPT1 are in part due to the effects of hypoxia-induced generation of reactive oxygen and nitrogen species (ROS/RNS). Results from these studies should provide information on new mechanisms by which hypoxia alters the critical signaling pathways and should elucidate some mechanisms of hypoxic pulmonary vasoconstriction. At birth, with the onset of breathing and oxygenation, both pulmonary arteries and veins must dilate to facilitate the dramatic fall in pulmonary vascular resistance and the increase in blood flow through the lungs. This allows for the onset of pulmonary gas exchange in the newly born infant. Any derangement in this process leads to hypoxemia in the newborn postnatally and a potentially life threatening situation of Persistent Pulmonary Hypertension of the Newborn (PPHN). Knowledge derived from research groups such as ours, will lead to a better understanding of the control of vasomotor tone in the fetal and neonatal pulmonary circulations and to treatment and prevention of pulmonary vascular disorders such as PPHN.